Post‐transcriptional control of expression of sFlt‐1, an endogenous inhibitor of vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a major modulator of angiogenesis. Biological effects of VEGF are mediated by endothelial cell‐surface receptors, KDR and Flt‐1. Alternative Flt‐1 RNA processing, involving retention of intron 13 and the use of intronic cleavage‐polyadenylation signals, produces a secreted form of Flt‐1, “sFlt‐1,” that binds VEGF with high affinity and can inhibit VEGF signaling. To probe mechanisms controlling sFlt‐1 expression, we have cloned and sequenced Flt‐1 intron 13 from a mouse genomic library and located RNA processing signals potentially involved in sFlt‐1 mRNA formation. A minigene construct containing Flt‐1 intron 13 directed the expression of both secreted (i.e., cleaved/polyadenylated) and transmembrane (i.e., spliced) forms of Flt‐1 mRNA and protein. Using rapid amplification of 3′ cDNA ends (3′‐RACE) and quantitative PCR (QPCR) analysis to test the activity of intronic cleavage‐polyadenylation signals, we observed that multiple sites were utilized for sFlt‐1 mRNA processing in both native tissues and Flt‐1 minigene transfectants. In transfectants, the most distal signal was utilized preferentially. The potential for interaction between pathways leading to sFlt‐1 or full‐length Flt‐1 was evaluated using QPCR to measure relevant mRNAs after transfection with signal mutants. Decreased expression of sFlt‐1 mRNA in cleavage‐polyadenylation mutants was accompanied by reciprocal increases in full‐length Flt‐1 mRNA. Multiple sFlt‐1 mRNA species are formed that differ by up to 3.9 kb in their 3′‐untranslated regions (UTRs), which contain sites of potential regulatory importance. The reciprocity between sFlt‐1 and Flt‐1 mRNA expression suggests a novel post‐transcriptional mechanism by which sFlt‐1 protein production and, thereby, responsiveness to VEGF, may be modulated. © 2004 Wiley‐Liss, Inc.